Human induced pluripotent stem cells (hiPSCs1–3) are useful in disease modeling and drug discovery, and they promise to provide a new generation of cell-based therapeutics. To date there has been no systematic evaluation of the most widely used techniques for generating integration-free hiPSCs. Here we compare Sendai-viral (SeV)4, episomal (Epi)5 and mRNA transfection mRNA6 methods using a number of criteria. All methods generated high-quality hiPSCs, but significant differences existed in aneuploidy rates, reprogramming efficiency, reliability and workload. We discuss the advantages and shortcomings of each approach, and present and review the results of a survey of a large number of human reprogramming laboratories on their independent experiences and preferences. Our analysis provides a valuable resource to inform the use of specific reprogramming methods for different laboratories and different applications, including clinical translation.

Post-translational modification (PTM) of proteins allows cells to regulate protein functions, transduce signals and respond to perturbations. PTMs expand protein functionality and diversity, which leads to increased proteome complexity. PTM crosstalk describes the combinatorial action of multiple PTMs on the same or on different proteins for higher order regulation. Here we review how recent advances in proteomic technologies, mass spectrometry instrumentation, and bioinformatics spurred the proteome-wide identification of PTM crosstalk through measurements of PTM sites. We provide an overview of the basic modes of PTM crosstalk, the proteomic methods to elucidate PTM crosstalk, and approaches that can inform about the functional consequences of PTM crosstalk.

mTORC2 phosphorylates AKT in a hydrophobic motif site that is a biomarker of insulin sensitivity. In brown adipocytes, mTORC2 regulates glucose and lipid metabolism, however the mechanism has been unclear because downstream AKT signaling appears unaffected by mTORC2 loss. Here, by applying immunoblotting, targeted phosphoproteomics and metabolite profiling, we identify ATP-citrate lyase (ACLY) as a distinctly mTORC2-sensitive AKT substrate in brown preadipocytes. mTORC2 appears dispensable for most other AKT actions examined, indicating a previously unappreciated selectivity in mTORC2-AKT signaling. Rescue experiments suggest brown preadipocytes require the mTORC2/AKT/ACLY pathway to induce PPAR-gamma and establish the epigenetic landscape during differentiation. Evidence in mature brown adipocytes also suggests mTORC2 acts through ACLY to increase carbohydrate response element binding protein (ChREBP) activity, histone acetylation, and gluco-lipogenic gene expression. Substrate utilization studies additionally implicate mTORC2 in promoting acetyl-CoA synthesis from acetate through acetyl-CoA synthetase 2 (ACSS2). These data suggest that a principal mTORC2 action is controlling nuclear-cytoplasmic acetyl-CoA synthesis.

Leigh syndrome is a fatal neurometabolic disorder caused by defects in mitochondrial function. mTOR inhibition with rapamycin attenuates disease progression in a mouse model of Leigh syndrome (Ndufs4 KO mouse); however, the mechanism of rescue is unknown. Here we identify PKC downregulation as a key event mediating the beneficial effects of rapamycin treatment of Ndufs4 KO mice. Assessing the impact of rapamycin on the brain proteome and phosphoproteome of Ndufs4 KO mice we find that rapamycin restores mitochondrial protein levels, inhibits signaling through both mTOR complexes, and reduces the abundance and activity of multiple protein kinase C (PKC) isoforms. Administration of PKC inhibitors increases survival, delays neurological deficits, prevents hair loss, and decreases inflammation in Ndufs4 KO mice. Thus, PKC may be a viable therapeutic target for treating severe mitochondrial disease. Reporting Summary Further information on research design is available in the Nature Research Reporting Summary linked to this article.